Difference between revisions of "Part:BBa K782022"

 
(2 intermediate revisions by 2 users not shown)
Line 2: Line 2:
 
<partinfo>BBa_K782022 short</partinfo>
 
<partinfo>BBa_K782022 short</partinfo>
  
TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011)
+
* TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011).
 +
* DNA binding sites for individual TAL effectors are indicated with square brackets [ ].
  
===Introduction===
+
 
Transcription activation like (TAL) effectors are DNA binding proteins with a high specifity, built from tandem repeats, with  near identical seqences, differing only in two amino acids in each repeat called “repeat variable diresidue” (RVD), which determine the specifity for a single nucleotide.(Scholze and Boch, 2011)
+
==Introduction==
 +
Transcription activation like (TAL) effectors are DNA binding proteins with a high specifity, built from tandem repeats, with  near identical seqences, differing only in two amino acids in each repeat called “repeat variable diresidue” (RVD), which determine the specifity for a single nucleotide.(Scholze and Boch, 2011).
  
 
We designed our construct with [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782072 12]  binding sites for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782005 TALD], upstream of a CMV promoter (constitutive promoter for expression in mammalian cells). Downstream of the promoter we cloned fLuciferase (firefly luciferase) to function as a reporter.
 
We designed our construct with [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782072 12]  binding sites for [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782005 TALD], upstream of a CMV promoter (constitutive promoter for expression in mammalian cells). Downstream of the promoter we cloned fLuciferase (firefly luciferase) to function as a reporter.
Line 11: Line 13:
 
[[Image:12×D_CMV_fLuc_shema1.png]]
 
[[Image:12×D_CMV_fLuc_shema1.png]]
  
'''Figure1:'''Schematic representation of our construct
+
'''Figure 1:'''Schematic representation of our construct.
  
===Characterisation===
+
==Characterisation==
 
HEK293T cells were transfected with the 12x[TALD] operator_CMV promoter_fLuciferase reporter and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782009 TALD:KRAB] (Figure 2). Tests showed, successful repression of fLuciferase with TAL repressor. Tests showed sucessful repression of the fLuciferase reporter (Figure 3).
 
HEK293T cells were transfected with the 12x[TALD] operator_CMV promoter_fLuciferase reporter and [https://parts.igem.org/wiki/index.php?title=Part:BBa_K782009 TALD:KRAB] (Figure 2). Tests showed, successful repression of fLuciferase with TAL repressor. Tests showed sucessful repression of the fLuciferase reporter (Figure 3).
  
Line 27: Line 29:
  
  
*fLuciferase was contributed by host lab
+
*fLuciferase was contributed by host lab.
*Binding sites for TAL effectors were ordered from IDT.
+
*Binding sites for TAL effectors were ordered from GeneArt.
 +
 
  
 
==References==
 
==References==
  
Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nature Biotechnology 29, 697–698
+
Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nature Biotechnology 29, 697–698.
  
Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53
+
Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53.
  
  

Latest revision as of 22:05, 26 September 2012

12x[TALD] operator_CMV promoter_fLuciferase

  • TALD label represents TAL effector 1295 from zebrafish experiments (Sander et al., 2011).
  • DNA binding sites for individual TAL effectors are indicated with square brackets [ ].


Introduction

Transcription activation like (TAL) effectors are DNA binding proteins with a high specifity, built from tandem repeats, with near identical seqences, differing only in two amino acids in each repeat called “repeat variable diresidue” (RVD), which determine the specifity for a single nucleotide.(Scholze and Boch, 2011).

We designed our construct with 12 binding sites for TALD, upstream of a CMV promoter (constitutive promoter for expression in mammalian cells). Downstream of the promoter we cloned fLuciferase (firefly luciferase) to function as a reporter.

12×D CMV fLuc shema1.png

Figure 1:Schematic representation of our construct.

Characterisation

HEK293T cells were transfected with the 12x[TALD] operator_CMV promoter_fLuciferase reporter and TALD:KRAB (Figure 2). Tests showed, successful repression of fLuciferase with TAL repressor. Tests showed sucessful repression of the fLuciferase reporter (Figure 3).

12×D CMV fLuc shema2.png

Figure 2:Schematic representation of repression experiments. A: in the absence of a TALD:KRAB, fLuciferase is constituitively expressed. B: when a TALD:KRAB is present, it binds to its binding site upstream of the CMV promoter and represses transcription of fLuciferase with the KRAB domain.


12×D CMV fLuc graf1.png

Figure 3:Testing repression of reporter luciferase gene. White column is showing constantly expressed reporter (fLuc), blue column is showing repression with TALD:KRAB.


  • fLuciferase was contributed by host lab.
  • Binding sites for TAL effectors were ordered from GeneArt.


References

Sander, J. D., Cade, L., Khayter, C., Reyon, D., Peterson, R. T., Joung, J. K., and Yeh, J.-R. J. (2011) Targeted gene disruption in somatic zebrafish cells using engineered TALENs. Nature Biotechnology 29, 697–698.

Scholze, H., and Boch, J. (2011) TAL effectors are remote controls for gene activation. Curr. Opin. Microbiol. 14, 47-53.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 659
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1371
    Illegal NgoMIV site found at 2715
    Illegal NgoMIV site found at 2736
    Illegal NgoMIV site found at 3051
    Illegal AgeI site found at 408
    Illegal AgeI site found at 640
    Illegal AgeI site found at 2439
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2621